Method for Preparation of N-Acetyl Cysteine Amide and Derivatives Thereof

ABSTRACT

The present invention includes methods for making and isolating N-acetylcysteine amide, intermediates and derivatives thereof comprising: contacting cystine with an alcohol and a chlorinating reagent to form an organic solution containing L-cystine dimethylester dihydrochloride; combining dried or undried L-cystine dimethylester dihydrochloride with a triethylamine, an acetic anhydride, and an acetonitrile to form a di-N-acetylcystine dimethylester; mixing dried di-N-acetylcystine dimethylester with ammonium hydroxide to form a di-N-acetylcystine amide; and separating dried di-N-acetylcystine dimethylester into N-acetylcysteine amide with dithiothreitol, triethylamine and an alcohol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/561,129, filed Sep. 20, 2017, the entire contents of which areincorporated herein by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of methods forpreparing N-Acetyl Cysteine Amide (N-acetylcysteine amide, NACA) andderivatives thereof.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with synthesis of N-Acetyl Cysteine Amide.

One such method is taught in U.S. Patent Publication No. 20170183302,filed by Warner, et al., entitled “Method for Preparation of N-AcetylCysteine Amide”. Briefly, these inventors teach an process for thepreparation of N-acetyl-L-cysteine amide (NACA) starting withN-acetyl-L-cysteine that involves contacting N-acetyl-L-cysteine with anorganic alcohol and an inorganic acid to form an organic solutioncontaining N-acetyl-L-cysteine ester; neutralizing the acid in theorganic solution with an aqueous solution containing a base to form aneutralized mixture; separating an organic solution containingN-acetyl-L-cysteine ester from the neutralized mixture; removing theN-acetyl-L-cysteine ester from the organic solution under reducedpressure; and contacting the N-acetyl-L-cysteine ester with ammonia.

Another such method for the preparation of N-acetyl cysteine amide(NACA) was previously described by Martin, et al., entitled “Amides ofN-Acylcysteines as Mucolytic Agents”, J. Med. Chem. 1967, 10, 1172-1176.

However, a need remains for developing an efficient method for theeffective, large-scale synthesis of N-acetyl cysteine amide thatprovides the product in high chemical yields and, in particular, highchemical and enantiomeric purity, without the need for chromatography.

SUMMARY OF THE INVENTION

This disclosure describes an efficient method or process for thepreparation of NACA in high chemical yields and high enantiomericpurity. Dimethyl 3,3′-disulfanediyl(2R,2′R)-bis(2-aminopropanoate)dihydrochloride is commonly referred to herein as L-cystine dimethylester dihydrochloride. Dimethyl3,3′-disulfanediyl(2R,2′R)-bis(2-acetamidopropanoate) is commonlyreferred to herein as di-N-acetylcystine dimethyl ester or Di-NACMe.(2R,2′R)-3,3′-disulfanediylbis(2-acetamidopropanamide) is commonlyreferred to herein as Di-NACA. (R)-2-acetamido-3-mercaptopropanamide iscommonly referred to herein as NACA or NPI-001 or N-acetylcysteineamide. Specifically, disclosed herein is a process comprising:contacting cystine with an alcohol and a chlorinating reagent to form anorganic solution containing L-cystine dimethylester dihydrochloride;combining dried or undried L-cystine dimethylester dihydrochloride witha triethylamine, an acetic anhydride, and an acetonitrile to form adi-N-acetylcystine dimethylester; mixing dried di-N-acetylcystinedimethylester with ammonium hydroxide to form a di-N-acetylcystineamide; and reducing dried di-N-acetylcystine dimethylester intoN-acetylcysteine amide with dithiothreitol, triethylamine and analcohol. In one aspect, the alcohol is an organic alcohol selected froman alkyl alcohol, methanol, ethanol, propanol, iso-propanol or butanol.In another aspect, the step of contacting L-cystine with an alcohol anda chlorinating reagent to form an organic solution containing L-cystinedimethylester dihydrochloride is conducted at −10 to 10° C. and thenheated to reflux at 65 to 70° C. to completion. In another aspect, thechlorinating agent is thionyl chloride. In another aspect, the furthercomprising a solvent exchange between the contacting and the combiningsteps. In another aspect, the step of combining is performed at −10 to10° C. In another aspect, a precipitate formed in the combining stepthat is filtered and washed with ethyl acetate before drying undervacuum. In another aspect, the step of combining uses at least 15volumes acetonitrile at −10 to 10° C. before adding at least 4equivalents of triethylamine followed by at least 2 equivalents ofacetic anhydride. In another aspect, the organic solvent is ethylacetate. In another aspect, the process further comprises drying theorganic solution removed from the neutralized mixture with a dryingagent. In another aspect, the process further comprises the step offree-basing the triethylamine from the di-N-acetylcystine dimethylesterwith saturated sodium bicarbonate after reaction completion. In anotheraspect, the ammonia is provided in the form of aqueous ammoniumhydroxide. In another aspect, the contacting of the N-acetyl-L-cystineester with ammonia is performed at room temperature. In another aspect,no metals are used in the reduction of di-N-acetylcystine dimethylesterinto N-acetylcysteine amide. In another aspect, the step of removing theorganics under reduced pressure is performed at about 45° C. or less. Inanother aspect, the step of removing the organics under reduced pressureis performed at about 35° C. or less. In another aspect, the step ofremoving the organics under reduced pressure is performed at about 30°C. or less. In another aspect, the step of removing the organics underreduced pressure is performed at about 45° C. In another aspect, theorganic solution removed from the neutralized mixture is filtered toremove solids. In another aspect, the one or more reducing agents isselected from triphenylphosphine, thioglycolic acid or dithiothreitoland the organic solvent is tetrahydrofuran (THF), dichloromethane (DCM),isopropanol (2-propanol), or ethanol.

In another embodiment, the present invention includes a compound havinga formula:

In another embodiment, the present invention includes a compound havinga formula:

In another embodiment, the present invention includes a compound havinga formula:

In yet another embodiment, the present invention includes a process formaking N-acetylcysteine amide comprising:

In another embodiment, the present invention includes a process formaking N-acetylcysteine amide comprising: contacting L-cystine with analcohol and a chlorinating reagent to form an organic solutioncontaining L-cystine dimethylester dihydrochloride; combining driedL-cystine dimethylester dihydrochloride with a triethylamine, an aceticanhydride, and an acetonitrile to form a di-N-acetylcystinedimethylester; mixing dried di-N-acetylcystine dimethylester withammonium hydroxide to form a di-N-acetylcystine amide; and reducingdried di-N-acetylcystine dimethylester into N-acetylcysteine amide withdithiothreitol, triethylamine, and an alcohol, wherein the reduction iswithout the presence of a metal. In one aspect, the one or more reducingagents is selected from tris (2-carboxyethyl)phosphine, thioglycolicacid, dithiothreitol, and the organic solvent is THF or dichloromethane(DCM), isopropanol, or ethanol, and the base is triethylamine.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 shows a basic chemical synthesis of the present invention.

FIG. 2A shows a view of NPI-001 (NACA) molecule ‘A’ without atomlabelling. All non-hydrogen atoms are shown with thermal ellipsoids setat the 50% probability level. Color code: Carbon, grey; H, white; O,red; S, yellow.

FIG. 2B shows a portion of NACA derived from starting materialL-Cystine.

FIG. 3 shows a view of unit cell an axis of NPI-001 containing completemolecules. All atoms are shown with thermal ellipsoids set at the 50%probability level. Color code: Carbon, grey; H, white; O, red; S,yellow.

FIG. 4 shows results of liquid chromatography with mass spectrometricdetection.

FIG. 5 shows Simulated (120 K) XRPD 2θ diffractogram of NPI-001.

FIG. 6 shows ¹H-NMR of NACA.

FIG. 7 shows ¹H-NMR assignments for NACA (¹H and ¹³C assignments arebased on analysis of the 1D ¹H NMR, ¹H-¹³C HSQC and ¹H-¹³C HMBCspectra.),

FIG. 8 shows ¹³C-NMR of NACA.

FIG. 9 shows ¹³C-NMR assignments for NACA. (¹H and ¹³C assignments arebased on analysis of the 1D ¹H-¹³C HSQC and ¹H-¹³C HMBC spectra).

FIG. 10 shows combined thermogravimetricy/differential scanningcalorimetry scans of NACA,

FIG. 11 shows fourier transform-infrared spectrum (FT-IR) of NACA.

FIG. 12 shows a Method I Chromatogram Showing NACA and all Intermediatesand Starting Material.

FIG. 13 is a representative Chromatogram of Method-II Showing B1 and B2.

FIG. 14 is a representative chromatogram showing separation of R-NACAand S-NACA.

FIG. 15 shows XRPD of NPI-001 Form 1, NPI-001/urea pattern 1 andNPI-001/urea pattern 1 Crash Cool.

FIG. 16 shows XRPD Diffractograms of NPI-001 Form 1, diNACA, SodiumMethoxide, NPI-001/sodium methoxide pattern 1 from methanol andNPI-001/sodium methoxide pattern 1 from ethanol.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not limit the invention, except as outlined in the claims.

The present invention is directed to novel methods for making N-AcetylCysteine Amide (NACA), and novel intermediates thereof. Moreparticularly, the present invention takes advantage of the novelintermediates to significantly, and surprisingly, increase theefficiency of preparing NACA in both a high chemical yield and a highenantiomeric purity. Below is the basic chemical structure of NACA.

The following procedures may be employed for the preparation of thecompound of the present invention. The starting materials and reagentsused in preparing these compounds are either available from commercialsuppliers such as the Aldrich Chemical Company (Milwaukee, Wis.), Bachem(Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methodswell known to a person of ordinary skill in the art, followingprocedures described in such references as Fieser and Fieser's Reagentsfor Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y.,1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supplements,Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, JohnWiley and Sons, New York, N.Y., 1991; March J.: Advanced OrganicChemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock:Comprehensive Organic Transformations, VCH Publishers, New York, 1989,relevant portions of which are incorporated herein by reference.

NACA is manufactured in 4 steps as shown in FIG. 1. The startingmaterial is the naturally occurring L-cystine with the L conformation onboth cysteine subunits. In the first step the two acid groups areprotected by forming the di-methyl ester of L-cystine. In the secondstep the two nitrogens on the L-cystine are reacted with aceticanhydride to give the di-NACME which is in essence L-cystine with bothacids protected as methyl esters and both primary amine groups protectedwith acetyl groups. Step 3 converts the methyl ester groups to primaryamides. Step 4reductively cleaves the Di-NACA intermediate to give NACA.

The preparation of NACA has one starting material that contributessignificantly to the backbone of the final structure: L-cystine. In FIG.2B, the portion of NACA that is in the dotted box is derived fromL-cystine. L-Cystine provides the chiral center and the amino acid core.Two common reagents, acetic anhydride and ammonium hydroxide, provideatoms by derivatizing the carboxylic acid and primary amine,respectively.

TABLE 1 List of Starting Materials Starting Material Quality OtherL-cystine ≥98.5% pure BSE/TSE- certification

Control of Starting Materials. Currently the quality of L-cystine iscontrolled based on the specification provided by the manufacturer andincoming testing performed by the Contract Manufacturing Organization(CMO). The release testing performed by CMO is shown in Table G. Thesupplier tests the L-cystine according to the USP monograph for thismaterial.

TABLE 2 L-Cystine Testing Attribute Test Method Specification/LimitAppearance Visual inspection White powder ID NMR (USP<761>; D2O)Conforms to structure Purity¹ Titration 98.5-101.0% Optical RotationVendor method −215 to −225° ¹A use test may be substituted for thepurity test

Reagents and Solvents. A list of reagents and solvents used in each stepof the manufacturing process is provided in Table H and Table I,respectively.

TABLE 3 List of Reagents Step Reagent Quality 1 Thionyl chloride ≥97% 2,4 Triethylamine ≥99% 2 Acetic anhydride ≥99% 2 Sodium bicarbonate ≥99% 2Sodium sulfate — (anhydrous) 3 Ammonium Hydroxide 28-32%  4Dithiothreitol ≥98%

TABLE 4 List of Solvents Step Solvent Quality 1 Methanol ≥99% 1, 4methyl t-Butyl Ether (MTBE) ≥99% 2 Acetonitrile ≥99% 2 Ethyl acetate≥99% 3, 4 89.0-92.0% EtOH (v/v) ≥99% EtOH + MeOH + iPrOH (v/v)

In the first step the methyl ester of L-cystine was formed. L-cystineand methanol were cooled to −10° C. before slowly adding thionylchloride. The skilled artisan will recognize methanol can be substitutedwith other similar reactive alcohols, and/or other alternativecarboxylic acid activating agents (e.g., oxalyl chloride, CDI, etc). Theslurry was then heated to reflux to produce a solution. Initially, thereaction was held for 4 hours before proceeding to the workup. UsingHPLC it was found to not achieve full conversion. Holding the reactionat reflux for 16 hours proved to be effective for conversion. Uponverifying reaction completion, the solution was solvent-exchanged intoethyl ether. Depending on the method of making, other such solvent mayalso be used, e.g., Methyl tert-butyl ether (MTBE). MTBE has proven tobe an effective replacement for ethyl ether, as such the solution can besolvent-exchanged successfully into MTBE, and can also be used forwashes. The white solids were filtered and washed with MTBE beforedrying at 45° C. under vacuum to a yield of 95-99% with a purity of96-99%.

In Step 2, the L-cystine dimethylester dichydrochloride is converted todi-N-acetylcystine dimethylester. L-cystine dimethylesterdihydrochloride, as a slurry in acetonitrile, is cooled to 0±5° C. Tothe cooled solution is first added 4.1 equivalents of triethylaminefollowed by 2.1 equivalents of acetic anhydride while maintaining aninternal temperature of ≤5° C. throughout the additions. After aging fornot less than 30 minutes reaction completion is confirmed by HPLC. Tothe reaction is added ethyl acetate and an aqueous workup is performed.Upon completion of the aqueous workup the organic solution is fullyexchanged into ethyl acetate by vacuum distillation of the acetonitrileand replacement with ethyl acetate several times. The product,di-N-acetylcystine dimethylester, is isolated by filtration and dried toa 73-75% yield with a purity of 93-97%. Step 2 can be accomplished in acouple of different ways. While not as efficient, the L-cystinedimethylester dihydrochloride was free-based using 300 weigh percentamberlyst-A21 in 10 volumes acetonitrile. The solution of free-basedmaterial in acetonitrile was then acetylated using 2.1 equivalentsacetic anhydride and 2.5 equivalents triethylamine at room temperaturefor 1 hour. The crude material was concentrated and dissolved into ethylacetate before being washed with saturated sodium bicarbonate and brinesolutions. The washed organics were concentrated to dryness for a 73-75%yield with a purity of 93-97%.

Alternatively, and more efficiently, the Step 2, triethylamine after thefree-basing was replaced with amberlyst-A21 for a total of 600 weightpercent. The acetonitrile was increased to 15 volumes to facilitateagitation. After free-basing is complete, the acetic anhydride can beadded at room temperature or below. Some impurities may be formed whenreaction was performed at room temperature, as such, the reaction can becooled to 0° C. to successfully reduce impurity formation. Afterreaction completion, the solution was previously concentrated todryness. Typically, filterable solids may be preferred in certainisolation steps. As such, the acetonitrile solution wassolvent-exchanged into ethyl acetate to yield filterable solids. Thesesolids were filtered and washed with ethyl acetate before drying at 45°C. under vacuum for a 45% yield with a purity of 98%. Thesolvent-exchanged material can also be chilled to 0° C. for 1 hour toincrease yield to 65% while achieving similar purity.

Typically, triethylamine is cheaper than amberlyst-A21, as such thereaction can also be accomplished using only triethylamine. This wasachieved by cooling the mixture of LL-cystine dimethylesterdihydrochloride in 15 volumes acetonitrile to 0° C. before adding 4.1equivalents of triethylamine followed by 2.1 equivalents of aceticanhydride. Previously, using amberlyst-A21 to free-base, thehydrochloride salt formed was bound to amberlyst-A21 and filtered off ofthe material to yield a clear colorless L-cystine dimethylestersolution. Using triethylamine as free base led to the formation oftriethylamine chloride salts. These were filtered off before salting outtriethylamine using 0.5 N HCl in 13% NaCl solution. At this point, thereaction was solvent-exchanged into ethyl acetate and filtered similarto previous methods. The alternative method resulted in 88-92% yieldwith 93-95% purity.

Using only triethylamine for step 2 led to some loss of product beforeacid treatment, ineffective removal of triethylamine using acid, anddegradation of material. When the triethylamine acetylation was firstperformed, solids were filtered before treating the solution. However,HPLC of the solids revealed the presence of desired product, Di-NACMe.Upon reaction completion, the slurry was now dissolved in 15 volumesethyl acetate before treatment to maximize Di-NACMe in solution.Initially, 0.5 N HCl in 13% brine solution was used to treat thereaction solution. ¹H-NMR results of acid-treated product revealedsignificant amounts of triethylamine were still present in step 2product. In addition to the ineffective removal of triethylamine, thepresence of acid in the material before drying degraded the materialinto a brown taffy when heat was applied. Since acid not only failed toremove triethylamine but also degraded material, acid was avoided.Saturated sodium bicarbonate replaced HCl to free-base triethylamine,acetic acid and HCl after reaction completion. After treating reactionsolution with base to neutralize HCl, the belief was that triethylaminecould be azeotroped with acetonitrile (ACN) while acetic acid stayedbehind in ethyl acetate after the solvent-exchange.

The amination of Di-NACMe into Di-NACA was performed in ammoniumhydroxide similar to the NAC method. Initially, the solid was chargedwith 8.44 equivalents of 28-30% aqueous ammonium hydroxide. The solidsdissolved in solution as Di-NACA precipitated. The slurry was agitatedfor 2 hours after solid formation before filtering and washing withminimal water. The solids were dried at 45° C. under vacuum. This methodresulted in 60-70% yield with 70% purity. A solvent-exchange intoethanol after reaction completion further increased yield withoutsacrificing purity. The higher purity of the Di-NACA can be used toachieve a higher yield in the final reduction step. Additionally,Di-NACA may be recrystallized from water to further enhance the purity.

The reduction of Di-NACA to NACA can be accomplished using tris(2-carboxyethyl)phosphine (TCEP), thioglycolic acid, and/ordithiothreitol (DTT). One reduction method uses 1.1 eq TCEP in 15volumes 10:1 THF:water which was heated to reflux. Reduction bythioglycolic acid was performed using 2.5 eq thioglycolic acid. Thereaction was attempted using THF and ethanol as solvents. THF reactionshad a of ˜42% with a purity of 4%. Ethanol reactions had a yield of 2%with purity of 30%. With the goal of using ethanol, amberlyst-A21 andtriethylamine were evaluated as bases to aid reduction. Amberlyst-A21was determined to be the more valuable a base with yields of 45% andpurity of 85%. Amberlyst-A21 also reduced reaction time from 2 days intriethylamine-mediated reactions to 3 hours in amberlyst-A21 mediatedreactions. Alternatively, DTT can also be used. Various possiblesolvents can be used, e.g., dichloromethane (DCM), methanol,isopropanol, ethanol and/or other alcoholic solvents. Ethanol was chosenfor its greater solubility of Di-NACA. The use of alternative reducingagents such as, but not limited to triphenylphosphine, TCEP,thioglycolic acid, etc., is contemplated herein. Solvents such as butnot limited to THF, EtOH, iPrOH, MeOH, DCM, water, etc. Bases whethercatalytic or stoichiometric such as but not limited to Et3N, NaOH,amberlyst-A21, etc. Initially, to effect the reduction of the disulfidebond, 1.5 equivalents triethylamine are added to Di-NACA dissolved inethanol followed by 1.5 equivalents of DTT. The solution was heated toreflux and held for 2 hours before solvent-exchanging into methyltert-butyl ether to precipitate filterable solids. Further optimizationof the reaction led to the reduction of triethylamine to a catalytic 0.1equivalents, DTT to 1.25 equivalents and the reaction temperature to62±3° C. with the effect of improving the impurity profile of theisolated NACA. This method yielded 85% of with a purity of ≥98.0%%. Thepurity of NACA may be further enhanced via recrystallization fromethanol.

These efforts to increase purity focused on the major impurities asmeasured by HPLC, namely, Di-NACA, DTT and cyclic DTT. NACA productionvia the NAC route revealed oxidation of NACA occurs when exposed to air.Ethanol and methyl t-Butyl Ether (MTBE) used in the reduction andsolvent-exchange were degassed in an effort to reduce Di-NACA. MTBEproved to be efficient in removing DTT and cyclic DTT, so an MTBEtrituration was performed to improve purity.

Di-NACA purifications were performed with the goal of taking purermaterial through reduction for purer NACA.

Step 1: Formation of L-Cystine Dimethylester Dihydrochloride

Weight/ Reagents/Materials MW Density Eqs. Moles Volume L-Cystine,≥98.5% 240.29 — 1.0  208  50 kg Thionyl Chloride, 118.97 1.64 2.41 504 60 kg ≥97% Methanol (MeOH),  32.04 0.79 12.5 vol — 625 L  ≥99%Methyl-tert Butyl Ether  88.15 0.74   8 vol — 400 L  (MTBE), ≥99%

Set-up: A 2000 L glass-lined reactor;

50 kg L-Cystine; and

625 L Methanol was charged to the reactor and agitated while cooling to−10° C.

60 kg Thionyl Chloride was slowly added at ≤−5° C. After additioncompletion, the reaction material was heated to reflux and held for 16hours. After the reaction was verified as complete by HPLC (≤0.5%starting material), the reaction was cooled to room temperature. Thereaction mixture was concentrated to 6 volumes before solvent-exchanginginto 3×8 volumes MTBE. The resulting slurry was agitated at roomtemperature for 1 hour before being filtered and washed with MTBE. Thesolids were dried at 45° C. under vacuum.

Yield: 68.15 kg (95.9%), Purity: 95.8%

Step 2: Formation of di-N-acetyl-1-cystine dimethylester (Di-NACMe)

Weight/ Reagents/Materials MW Density Eqs. Moles Volume L-CystineDimethylester Dihydrochloride, 341.26 — 1.0  76.2  26 ≥95% Acetonitrile,≥99%  41.05 0.79 23 vo1 — 472 Triethylamine (TEA), ≥99% 101.19 0.73 4.2316.2  32 Acetic Anhydride, ≥99% 102.09 2.1 156.7  16 Ethyl Acetate,≥99% 88.1 41 vol — 958

Set-up: A 800 L glass lined reactor

26 kg L-Cystine Dimethylester Dihydrochloride and

390 L Acetonitrile (ACN) is charged to the reactor and agitated whilecooling to 0° C.

32 kg Triethylamine is added to the reactor at ≤5° C.

16 kg Acetic Anhydride is slowly added to the reactor at ≤5° C. Uponaddition completion the reaction is held for 30 minutes at 5±5° C. Afterreaction was verified as complete by HPLC (≤0.5% starting material), 10volumes ethyl acetate was charged to the reactor and agitated toambient. The resulting reaction mixture was washed with 2×2 volumessaturated bicarbonate. The aqueous layer was back extracted with 5volumes ethyl acetate. All organics were combined and dried over sodiumsulfate and polish filtered. The filtrate was concentrated to 5 volumesbefore azeodrying with 2×4 volumes acetonitrile followed by asolvent-exchange into 4×6 volumes ethyl acetate. The resulting slurrywas agitated at 0° C. for 1 hour before being filtered and washed with 2volumes ethyl acetate. The solids were dried at 25° C. under vacuum.

Average Yield: 29.56 kg (100%), Average Purity: 92.2%

Step 3: Formation of Di-NACA

Weight/ Reagents/Materials MW Density Eqs. Moles Volume Di-NACMe 352.42— 1.0   247 87.05 kg 28-30% Ammonium Hydroxide  35.05 0.9  8.44 2088  244 kg Ethanol, absolute 200 proof  46.07 0.79 17 vol — 1483 L 

Set-up: A 800 L glass lined reactor

244 kg Degassed 28-30% NH4OH (aq) was cooled to 0° C. before

87.05 kg Di-NACMe was charged to the reactor and agitated for 4 hours.After reaction was verified as complete by HPLC (≤0.5% startingmaterial), the reaction mixture was solvent-exchanged into 3×5 volumesdegassed ethanol. The resulting slurry was agitated at 0° C. for 30minutes before being filtered and washed with cold degassed ethanol. Thesolids were dried at 45° C. under vacuum.

Average Yield: 52 kg (67.1%), Average Purity: 72.0%

Step 3A: Purification of Di-NACA

Weight∧ Reagents/Materials MW Density Eqs. Moles Volume Di-NACA 322.40 —1.0 161  52 kg Process Water, Filtered  18.02 1.0 8 vol — 416 L 

Set-up: A 800 L glass lined reactor

52 kg Di-NACA and

416 L Degassed Water were charged to the flask and agitated whileheating to reflux.

Upon dissolution, the reaction solution was allowed to cool overnight.The solids were filtered and washed with 2 volumes cold degassed water.The solids were dried at 45° C. under vacuum.

Recovery: 36 g (69%), Purity: 86.4%

Step 4: Formation of NACA

Weight/ Reagents/Materials MW Density Eqs. Moles Volume Di-NACA 322.40 —1.0 112 36 kg Ethanol, absolute, 200 proof  46.07 0.79 20 vo1 — 720 L  Triethylamine (TEA), ≥99% 101.19 0.73 0.1 8.8 1.1 kg  Dithiothreitol,≥98% 154.25 —  1.25 143 22 kg (aka 1,4-Dithiothreitol and DL-Dithiothreitol)

Set-up: A 800 L glass lined reactor

720 L Degassed Ethanol,

1.1 kg Triethylamine,

22 kg Dithiothreitol and

36 kg of Di-NACA were charged to the reactor before heating the reactionto 62±3° C. The reaction is held at temp for 2 hours. After reaction wasverified as complete by HPLC (≤0.5% starting material on overloadedcolumn), cool reaction to ambient. The reaction solution was polishfiltered and concentrated to 10 volumes before solvent exchanging into4×10 volumes degassed MTBE. The resulting slurry was agitated overnightbefore being filtered and washed with 2 volumes degassed MTBE. Thesolids were dried @45° C. under vacuum.

Yield: 27.8 kg (77.2%), Purity: 98.5%

FIG. 1 shows a basic chemical synthesis of the present invention. FIG.2A shows a view of NPI-001 molecule ‘A’ without atom labeling. Allnon-hydrogen atoms are shown with thermal ellipsoids set at the 50%probability level. Color code: Carbon, grey; H, white; O, red; S,yellow. FIG. 2B shows a portion of NACA derived from starting materialL-Cystine. FIG. 3 shows a view of unit cell an axis of NPI-001containing complete molecules. All atoms are shown with thermalellipsoids set at the 50% probability level. Color code: Carbon, grey;H, white; O, red; S, yellow. FIG. 4 shows results of liquidchromatography with mass spectrometric detection. FIG. 5 shows Simulated(120 K) XRPD 2θ diffractogram of NACA. FIG. 6 shows ¹H-NMR of NACA. FIG.7 shows ¹H-NMR assignments for NACA (¹H and ¹³C assignments are based onanalysis of the 1D ¹H NMR, ¹H-¹³C HSQC and ¹H-¹³C HMBC spectra.). FIG. 8shows ¹³C-NMR of NACA. FIG. 9 shows ¹³C-NMR assignments for NACA. (¹Hand ¹³C assignments are based on analysis of the 1D ¹H NMR, ¹H-¹³C HSQCand ¹H-¹³C HMBC spectra). FIG. 10 shows combinedthermogravimetricy/differential scanning calorimetry scans of NACA. FIG.11 shows fourier transform-infrared spectrum (FT-IR) of NACA.

Analytical procedures for NACA. The analytical methods for the testingof NACA drug substance are listed in Table 5. Most of the methods areUSP compendial tests. The additional NACA drug substance methods (whichare not compendial) include two HPLC methods for assay and impurities,and one chiral HPLC method for chiral purity. Each of the non-compendialmethods is described in more detail in sections that follow.

TABLE 5 List of Analytical Procedures for NACA Drug Substance Test TestMethod Description Appearance Visual A sample of the solid material isexamined visually for form and color. ID-1 IR Method follows USP<197A>ID-2 ¹H-NMR Method follows USP<761> Potency/ Calculated Purity = (100 −% HPLC impurities − Assigned % H₂O − % ROI − % Total Residual PuritySolvents) Organic HPLC-Method I Reverse phase gradient HPLC method.Impurities/ HPLC Method II Reverse phase gradient HPLC method. RelatedSubstances Chirality Optical Rotation Method follows USP<781> (c 1.00,MeOH) Chiral Purity Chiral HPLC Chiral HPLC method Residue on USP<281>Method follows USP<281> Ignition DSC USP<891> Method follows USP<891>X-ray USP<941> Method follows USP<941> Powder Diffraction Heavy USP<233>Method follows USP<233> Metals Residual GC Method follows USP<467>Solvents (USP<467>) Water Karl Fischer Method follows USP<921> version1c Content Microbial Microbial Method follows USP<61> Limits enumerationTest for Method follows USP<62> specified organisms

HPLC Method for Purity and Related Substances. Analysis of the NACA drugsubstance for purity and related substances makes use of a reverse phaseHPLC with an ultraviolet (UV) detector. The method is summarized inTable 6. This method is also used as the in-process method for each stepto follow completion of reaction.

TABLE 6 Summary of NACA HPLC Method I (Purity and Related Substances)Method Element Description Column Phenomenex Synergi Hydro-RP, 4.6 × 250mm, 4 μm Detection 214 nm Column 40° C. Temperature Injection Volume 25μL Flow Rate 1.0 mL/minute Mobile Phase A 0.02% H₃PO₄ in H₂O MobilePhase B Acetonitrile (ACN) Time (min) % A % B Gradient  0.0 100 0 15.090 10 25.0 0 100 30.0 0 100 30.1 a 100 0 35.0 a 100 0 SystemSuitability 1. Specificity: No significant interference in the    blankchromatogram at retention times of    interest. 2. The S/N of the NACApeak in the 0.03% sensitivity    solution must be ≥10 3. The % RSD ofthe RT and peak area of NACA    in the 5 injections of the first samplemust    be ≤2.0%. 4. The recovery of one standard prep (average of all   injections) versus a second standard prep    (average of allinjection) must be 100.0 ± 2.0%. a Equilibration time - no integration

FIG. 12 shows a Method I Chromatogram Showing NACA and all Intermediatesand Starting Material.

HPLC Method for Impurities B1 and B2. Method-I did not always detectimpurities B1 and B2. A second method, Method-II, was developed toreliably quantitate these two impurities. The method is summarized inTable 7. A representative chromatogram showing the retention times of B1and B2 is presented in FIG. 13.

TABLE 7 Summary of NACA HPLC Method II (Impurities B1 and B2) MethodElement Description Column Agilent Zorbax SB-Aq, 4.6 × 250 mm, 5 μmDetection 214 nm Column Temperature 40° C. Injection Volume 25 μL FlowRate 1.0 mL/minute Mobile Phase A 0.02% H₃PO₄ in H₂O Mobile Phase BAcetonitrile (ACN) Time (min) % A % B Gradient 0.0 100 0 5.0 100 0 20.00 100 25.0 0 100 25.01 100 0 35.0 100 0 System Suitability 1.Specificity: No significant interferences in the    blank chromatogramat retention times of    interest. 2. The S/N of the NACA peak in the0.03%    sensitivity solution must be ≥10 3. The % RSD of the RT andpeak area of NACA    in the 5 injections of the first sample must    be≤2.0%.

FIG. 13 is a representative Chromatogram of Method-II Showing B1 and B2

Chiral HPLC Method for Measuring Chiral Purity of NACA. A chiral methodwas developed to assess the chiral purity of NACA. The method issummarized in Table 8.

TABLE 8 Summary of Chiral HPLC Method for NACA Method ElementDescription Column Diacel Chiralpak IC-3, 4.6 × 150 mm Detection 217 nmColumn Temperature 35° C. Injection Volume 20 μL Flow Rate 0.8 mL/minuteMobile Phase 0.05% H₃PO₄ in 1:1 n-hexane:IPA (isocratic) SystemSuitability 1. Specificity: No significant interferences in the    blankchromatogram at retention times of    interest. 2. The resolutionbetween the enantiomers is    sufficient to allow for accurateintegration of    both peaks. 3. The S/N of the NACA peak in the 0.03%   sensitivity solution must be ≥10 4. The % RSD of the RT and peak areaof NACA    in the 6 injections of the first sample must be    ≤2.0%.

FIG. 14 is a chromatogram Showing Separation of R-NACA and S-NACA.

Preparation of D-NACA. D-Cystine was obtained from. D-Cystine wasdissolved in water and pH was adjusted to pH 9-10 with NaOH. Aceticanhydride was added dropwise at 0° C. and pH maintained at 9-10.Solution was stirred for 4 hours afterwhihcafter which it was acidifiedto pH ˜2 with HCl. The solvent was evaporated under reduced pressure. 20mL MeOH was added to the flask to dissolve contents. Solution wasfiltered. Filtrate was concentrated and evaporated. Thin layerchromoatrophy (MeOH:DCM:HOAc 1:8:1) indicated loss of starting materialand appearance of a single new peak for D-acetylcystine. D-Acetylcystinewas charged to a round-bottomed flask with 50 mL MeOH to which was added0.35 mL concentrated H₂SO₄ via syringe, dropwise, at ambienttemperature, with agitation. Solution turned turbid. IPC by TLC(MeOH:DCM, 1:9] showed no starting material. Solvent was evaporated.Fraction was neutralized with NaHCO3 (saturated), extracted with DCM (50ml twice), washed with water, dried over Na2SO4, filtered, purged withnitrogen and concentrated under reduced pressure to yield a net weightof 7.8 g white solid, formed in the refrigerator. N-acetylcysteinemethyl ester was charged into a 3-necked, round-bottomed flask withnitrogen bublet, and magnetic stirrer. Ammonium hydroxide at ambienttemperature was added and purged with nitrogen through reaction mixtureand agitated at ambient temperature. Solvent was evaporated under vacuumand a white solid formed. Ethanol was added and heated to form clearsolution and left to stand overnight. White solid crystallized, wasfiltered, washed with ethanol and purified by column chromatography.

Oral Solution of NACA.

A study was conducted to determine the solubilization of NACA inORA-SWEET® (Ora-Sweet is a commercially available syrup vehiclecontaining water, sucrose, glycerin, sorbitol, flavoring, bufferingagents (citric acid and/or sodium phosphate), methyl paraben andpotassium sorbate, pH 4.2 manufactured by Paddock Laboratories, Inc.,Minneapolis, Minn.). HPLC equipment and glass containers and stirrerswere utilized.

NACA at 80 mg/ml did not readily dissolve in ORA-SWEET, rather itrequired stirring for 20-30 minutes to achieve a solution. However, NACAwas readily dissolved in water with shaking for 30 seconds, followed bydilution with an equal volume of ORA-SWEET with shaking for 20-30seconds. Therefore, NACA was dissolved in 50 mL water followed by 50 mLOra-Sweet, shaked to dissolve in an opaque plastic bottle with closureand provided to subject for self-administration (oral ingestion).

NACA oral solution was also be prepared as 100 ml oral solution inORA-SWEET. Doses can be achieved ranged from 250 mg to 4000mg/day/patient. The NACA dissolution in water followed by dilution withORA-SWEET was found optimal for compounding. ORA-SWEET is a pale pinksolution with a cherry syrup flavor. NACA has a mild sulfur odor and abitter taste (like burned sesame seeds). When dissolved in ORA-SWEET,the odor and taste were masked.

The following instructions for preparation of NACA Oral Solution weredeveloped:

Weigh NACA [either 250, 750, 1500, 3000 or 4000 mg (±1 mg), asappropriate for the particular dose group] and place into a 125 mL(approximately) capacity opaque high density polyethylene, labeled (seeTable 9) bottle with opaque polypropylene screw cap.

TABLE 9 Container/closure for NACA Oral Solution Used for Phase 1 StudyComponent Description Bottle 125 mL opaque white high densitypolyethylene bottle Cap Polypropylene opaque white cap Label Pharmacyapproved label

Measure 50 mL of Purified Water and pour into each bottle containingNACA and shake vigorously by hand (at least 30 seconds) to dissolve.

Measure 50 mL Ora-Sweet and pour into each bottle containing NACA andshake vigorously by hand (at least 30 seconds) to dissolve.

A subject drinks entire solution, followed by two 20-ml rinses of thecontainer with water, which are also drank.

Tablet or Capsule of NACA for clinical phase 1 or clinical phase IItrials:

The qualitative composition for NACA Tablets is presented in Table 10.

TABLE 10 Qualitative Composition of NACA Tablets Component QualityStandard NACA Nacuity Lactose NF Microcrystalline Cellulose NFCroscarmellose Sodium NF Stearic acid NF

NACA Tablets, 250 mg, are formulated as an immediate release drugproduct. A roller-compacted blend containing 250 mg NACA is compressedinto round, biconvex tablets.

The formulation use to manufacture NACA Tablets is a roller compactedblend of common excipients (Table 11). The blend is compressed intoround, biconvex shaped tablets.

TABLE 11 Quantitative Composition of NACA Tablets Quality Componentmg/tablet weight % Function Standard NACA 250.0 50 Drug Nacuitysubstance Lactose 42.875 8.575 Filler NF Microcrystalline 177.125 35.425Filler NF Cellulose Croscarmellose 25 5.0 Disintegrant NF Sodium Stearicacid 5 1.0 Lubricant NF TABLET WT 500 100 — —

Type of Container and Closure for Dosage Form

NACA Tablets, 250 mg, are packaged in high density polyethylene (HDPE)bottles with foil induction seal and a white polyproplylene screw-topclosure. Excipients used for the formulation meet compendial standards.Lactose functions as a filler. Microcrystalline cellulose functions as afiller. Croscarmellose Sodium functions as a disintegrant. Stearic Acidfunctions as a lubricant. Excipients were screened by assessing thestability of NACA in mixtures with each excipient. Mixtures of NACA witheither microcrystalline cellulose, lactose monohydrate, croscarmellosesodium and crospovidone/Kollidon CL and hydroxypropyl celluloseexhibited less degradation of NACA compared to other excipients (Table12). Hydroxypropyl cellulose exhibited greater levels of impurities thanthe other lead excipients (data not shown). Based on these results aswell as the collective experience of the Formulators, microcrystallinecellulose, lactose monohydrate, croscarmellose sodium and steric acidwere chosen as excipients for NACA Tablets.

TABLE 12 Stability results for NACA/excipient mixtures after 4 weeks at40° C./75% RH Sample ID Excipient Ratio of API:Excipient % Assay SPt:17004Q4: P NA 1:0 95.3 SPL-1700405-P Microcrystalline Cellulose PH 1021:1 94.9 SPL-1700406-P Lactose Monohydrate (Fastflo) 1:1 93.2SPL-1700407-P Croscarmellose Sodium 4:1 93.6 SPL-1700408-PCrospovidone/Kollidon CL 4:1 94.4 SPl-1700409-P Sodium Lauryl Sulfate4:1 26.2 SPL-1700410-P Colloidal Silicon Dioxide 4:1 92.7 SPL-1700411-PMagnesium Stearate 9:1 77.5 SPL-1700412-P Sodium Stearyl Fumarate 9:186.7

Tables 13 and 14 were prepared. The use of roller compaction offormulations of NACA with various excipients yielded powders withacceptable flow properties. Roller compaction followed by compressionyielded tablets with acceptable properties based on friability andhardness. The clinical formulation was selected based on acceptable2-week stability data.

Dry blending of formulations of NACA with various excipients yieldedpoorly flowing powders. Dry blending followed by compression yieldedtablets that were unsatisfactory based on friability and hardness.

The use of roller compaction of formulations of NACA with variousexcipients yielded powders with acceptable flow properties. Rollercompaction followed by compression yielded tablets with acceptableproperties based on friability and hardness.

TABLE 13 Finished Prototype NACA Tablets by Roller Compaction PackagingBatch Formulation Product Batch # Configurations* NAC1G50%0401NAC1T250mg0401A 1 Low Roller Compaction Force NAC1T250mg0401 B 2 (3 kN)NAC1G50%0402 NAC1T250mg0402A 1 High Roller Compaction NAC1T250mg0402B 2Force (6 kN) NAC1G50%0501 NAC1T250mg0501A 1 Low Roller Compaction ForceNAC1T250mg0501 B 2 (3 kN) NAC1G50%0502 NAC1T250mg0502A 1 High RollerCompaction NAC1T250mg0502B 2 Force (6 kN) *Packaging Configuration 1(With Desiccant): Bottle: 60CC 33/400 W-HDPE ROUND BTL Cap: 33MM SECURXwith SG-75M liner Desiccant: DESICCANT CANISTER SILICA GEL 2GM Fill: 20tablets per bottle *Packaging Configuration 2 (Without Desiccant):Bottle: 60CC 33/400 W-HDPE ROUND BTL Cap: 33MM SECURX with SG-75M linerFill: 20 tablets per bottle

TABLE 14 NACA Tablet Prototype Batch Formulations by Roller CompactionNAC1G50%0401 NAC1G50%0402 NAC1G50%0501 NAC1G50%0502 mg/tablet weightmg/tablet weight mg/tablet weight mg/tablet weight Low Force High ForceLow Force High Force Component g % g % g % g % INTRAGRANULAR NACA 750 50750 50 750 50 0 0 NACA Direct Blend 70% 0 0 0 0 0 0 0 71.42 (NAC1B70%01)Lactose 330 22 330 22 128.6 8.57 857.0 0 Microcrystalline Cellulose 33022 330 22 128.6 8.57 0 0 Croscarmellose Sodium 75 5 75 5 53.6 3.57 0 0Stearic Acid 7.5 0.5 7.5 0.5 10.6 0.71 0 0 EXTRAGRANULARMicrocrystalline Cellulose 0 0 0 0 402.9 26.86 322.3 26.86Croscarmellose Sodium 0 0 0 0 21.4 1.43 17.2 1.43 Stearic Acid 7.5 0.57.5 0.5 4.3 0.29 3.5 0.29 Total 1500 100 1500 100 1500 100 1200 100*NAC1B70%01 = 70% NACA + 12% Lactose + 12MCC + 5% CroscarmelloseSodium + 1% Stearic Acid

NACA Tablet Dry Blend Formulation Development

NACA Tablet formulations (Table 15) were dry blended and directlycompressed. The flow of formulation from the hopper to the press was notuniform. Also, the resulting tablets suffered poor friability, hardnessand capping. As a result dry blending was abandoned.

TABLE 15 Composition of Prototype NACA Tablet Dry Blend FormulationsPrototype Batch Composition Batch Batch NAC1B50%0101 NAC1B50%201Component g % g % NACA 600 50 600 50 Lactose 528 44 — — MicrocrystallineCellulose — — 528 44 Croscarmellose Sodium 60 5 60 5 Stearic Acid 12 112 1 Total 1200 100 1200 100

The formulations described above can also be used as a dry blend forfilling into capsules.

The relation between micronization conditions and an initial increase ofthe degradation product DiNACA was further investigated. Comparingstainless steel and zirconium oxide grinding jars gives a clearindication that steel samples undergo a time dependent increase of thedegradation product DiNACA. In contrast, using zirconium oxide jars andballs did not lead to an initial increase of the degradation productDiNACA. Therefore the zirconium oxide milling process was furtherinvestigated and successfully optimized regarding particle sizedistribution, milling parameters and impurity profile for the 1% NACAformulation. Zirconium oxide milling process was investigated andsuccessfully optimized regarding particle size distribution, millingparameters and impurity profile for the 1% NACA formulation. Theparticle size distribution by laser diffraction analysis was x₁₀=1.3 μm,x₅₀=4.7 μm and x₉₀=12.3 μm. Batches were prepared and found to be stablefor up to 4 weeks at ambient temperature.

Single Crystal X-Ray Diffraction.

The absolute structure of NACA has been determined by single crystalX-ray diffraction from suitable crystals grown from cooling of asaturated 2-propanol NACA solution to ambient conditions. Single crystalanalysis of crystals clearly shows that the material is NACA with theexpected bond connectivity. The absolute stereochemistry has been provedin the crystal with excellent confidence, as confirmed by the Flackparameter being −0.02(3). The density of the material is high, reducingthe risk that a more stable polymorph is even possible and the hydrogenbonding network observed satisfies the expected functionality observedin NACA. The predicted XRPD from the SC-XRD data is consistent with theForm 1 material, indicating that the crystal was representative of thebulk material. Data was collected and found to be twinned, therefore,was refined accordingly using HKLF5 and BASF commands, locating a twocomponent twin with BASF scales 0.7271(10):0.2729(10) in the Monoclinicspace group P21 where two complete formula units of NACA were found inthe asymmetric unit only. No disorder was noted in the final structurewith final a R1 [I>2σ(I)] of 3.30% obtained with Flack parameter of−0.02 with e.s.d 0.03 determined using 1549 quotients that is suitableto accurately determine the IUPAC name of NACA as2R)-2-(acetylamino)-3-sulfanylpropanamide (=N-acetyl-L-cysteineamide=(R)-2-acetylamino)-3-mercapto-propamide).

NACA, Form 1 overall structure quality factor: 1

Where:

1. Strong data set, no disorder, R1 ˜4%. Publishable quality.

2. Good data set, contains some minor disorder, R1 ˜6%. Publishablequality.

3. Average data set and/or easily modelled disorder or twinning.Publishable with care.

4. Weak data and/or major disorder or twinning that is not easilymodelled. Publishable in some cases.

5. Very weak data and/or unexplained features of data or model. Not ofpublishable quality.

Polymorphism.

A detailed polymorph screen of NACA (NACA) (NACA) has been performedusing a variety of solvents and experimental conditions. During theprimary screen, the most common solid form observed was pattern 1 (Table16).

TABLE 16 Crystallographic parameters and refinement indicators of NACA,Form 1. NACA, Form 1 Empirical formula C₅H₁₀N₂O₂S Formula weight  162.21Temperature/K  120(1) Crystal system Monoclinic Space group P2₁ a/Å  7.2832(2) b/Å   7.5542(2) c/Å  13.9686(4) α/°  90 β/°  98.6983(15) γ/° 90 Volume/Å³  759.70(4) Z, Z′ 4, 2 ρ_(calc) g/cm³   1.418 μ/mm⁻¹  0.369 F(000)  344.0 Crystal size/mm³ 0.384 × 0.207 × 0.131 RadiationMoKα (λ = 0.71073) 2Θ range for data 2.95 to 56.582 collection/° Indexranges −9 ≤ h ≤ 9, −10 ≤ k ≤ 10, −18 ≤ 1 ≤ 18 Reflections collected 5810Independent reflections 5810 [R_(int) = ?, R_(sigma) = 0.0288]Data/restraints/parameters 5810/1/186 Goodness of Fit   1.057 Final Rindexes [I > 2σ (I)] R₁ = 0.0330, wR₂ = 0.0869 Final R indexes [alldata] R₁ = 0.0350, wR₂ = 0.0900 Δρmax, Δρmin/e Å⁻³ 0.47/−0.53 FlackParameter  −0.02(3) R₁ = (Σ|F_(o)| − |F_(c)|)/Σ|F_(o)|); wR₂ = {Σ[w(F_(o) ² − F_(c) ²)²]/Σ [w(F_(o) ²)²]}^(1/) ² ; S = {Σ [w(F_(o) ² −F_(c) ²)²]/(n − p)}^(1/) ^(2.)

Several experiments yielded diffractogram patterns that were differentor, more commonly, had extra peaks observed. The extra peaks wouldindicate the presence of another form, albeit not in a pure phase.

Attempts to reproduce these forms failed using both crash cooling,evaporation and anti-solvent addition. Analysis of these attempts by NMRshowed that the material was still predominately the NACA material andthat it had not oxidized to Di-NACA. The lack of reproducibility ofthese potential forms is good evidence for their lack of stability. Thisstudy has clearly demonstrated that the NACA material exists as Form 1and that other forms are difficult, if not impossible, to produce.

Approximately 80 mg of NACA was weighed into each of 24 vials. Thesolvents listed below were added to the appropriate vials. Thequantities added were calculated (based on solubility studies) todissolve approx. 60% of the material. These mixtures were temperaturecycled between ambient and 40° C., in 4 hr cycles, for 72 hrs. Solidsisolated from the slurries are tested by XRPD. The resulting saturatedsolutions were separated into three separated vials for crash cooling,anti-solvent addition and evaporation experiments.

TABLE 17 List of Solvents Used in the Primary Polymorph Screen Solvent 1Acetone 2 Acetone/water (80:20) 3 Acetone/Heptane (75:25) 4 Acetonitrile5 Acetonitrile/water (80:20) 6 1-Butanol 7 1,2-Dimethoxyethane 81,4-Dioxane 9 Dioxane/water (80:20) 10 Ethanol 11 Ethanol/water (80:20)12 Ethanol/heptane (75:25) 13 Ethyl Formate 14 Isopropyl acetate 15Ethyl acetate 16 Methanol 17 Methanol/water (80:20) 18 Methyl Ethylketone 19 Nitromethane 20 1-Propanol 21 2-Propanol 22 2-Propanol/water(80:20) 23 Tetrahydrofuran 24 Water

Liquid Chromatography with Mass Spectrometric Detection

Column Temperature: 30° C.

Mobile Phase A: 0.1% v/v Formic in Water

Mobile Phase B: 0.1% v/v Formic in Acetonitrile

Diluent: Mobile Phase A: 50:50 Water: Acetonitrile

Flow Rate: 1.0 mL/min

Runtime: 25 minutes

Injection Volume: 10 μL

Detection: 190-400 nm

Gradient::

Time (minutes) Solvent B [%] 0 0 12 10 15 100 15.1 100 25 0

Instrument: LCQ Advantage Ion Trap MS

Sample concentration: 1 mg/ml, +ve ion mode by infusion

Source voltage (kV): 4.50

Source current (μA): 80.00

Sheath gas flow rate: 20.00

Aux/Sweep gas flow rate: 0.00

Capillary voltage (V): 8.00

Capillary temp (° C.): 200

Tube lens (V, Sp): 40.00

NACA/Urea Co-crystal. A primary co-crystal screen was conducted where 28potential co-crystal formers (CCFs) were screened (Table 18) in 6solvent systems under 2 process relevant crystallization conditions,namely, thermal maturation and evaporation. A NACA/urea co-crystal wasidentified (FIG. 15). The NACA/urea pattern 1 material was successfullyscaled up from four solvents as a part of the secondary co-crystalscreen, then fully characterized where it was found to be crystalline byXPRD and PLM with the expected XRPD pattern, thermally stable with highpurity. NMR analysis confirmed an approximate stoichiometric content ofurea contained within the material. The material appeared to be stableunder ambient conditions and elevated temperature (80° C.) but unstablewhen stored at high humidity for prolonged periods, showing degradationto the di-NACA. No signs of dissociation were identified in organicsolvents and solvent/water mixtures with low water activity but wasfound to dissociate in deionized water and solvent/water mixtures with ahigh water activity. An additional DSC experiment with post-XRPDanalysis confirmed that the exothermic event observed during the DSCcooling cycle is a recrystallization to NACA.

TABLE 18 Primary Co-Crystal Screen Co-Former List Co-Former GRAS 12-Picolinamide 2 5-Methylfurfural 3 5-Methylfurfurylamine 4 Adenine 5Citric Acid 6 Glycine 7 Hippuric Acid 8 L-Aspartic Acid 9 L-Proline 10L-Tyrosine 11 Malonic Acid 12 Melamine 13 Oxalic Acid 14 Theophylline 15Tromethamine 16 Urea 17 Xanthine 18 3,4-Dihydroxybenzoic Acid 19Camphoric Acid 20 Cytosine 21 Formamide 22 L-Cysteine 23 L-Methionine 24L-Serine 25 Threonine 26 Di-NACA 27 N-Acetyl-L-cysteine 28 Succinic acid

NACA Sodium Salt. A salt screen was conducted using 6 solvent systemsunder 2 process-relevant crystallization conditions, namely thermalmaturation and evaporation. From this study, one sodium salt wasidentified. The salt was formed using sodium methoxide from eitheracetonitrile, ethanol, methanol, or tetrahydrofuran as shown in FIG. 16.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In embodiments of any of the compositions andmethods provided herein, “comprising” may be replaced with “consistingessentially of” or “consisting of”. As used herein, the phrase“consisting essentially of” requires the specified integer(s) or stepsas well as those that do not materially affect the character or functionof the claimed invention. As used herein, the term “consisting” is usedto indicate the presence of the recited integer (e.g., a feature, anelement, a characteristic, a property, a method/process step or alimitation) or group of integers (e.g., feature(s), element(s),characteristic(s), property(ies), method/process steps or limitation(s))only.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skill in the art recognize themodified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A process for making N-acetylcysteine amidecomprising: contacting cystine with an alcohol and a chlorinatingreagent to form an organic solution containing L-cystine dimethylesterdihydrochloride; combining dried or undried L-cystine dimethylesterdihydrochloride with a triethylamine, an acetic anhydride, and anacetonitrile to form a di-N-acetylcystine dimethylester; mixing drieddi-N-acetylcystine dimethylester with ammonium hydroxide to form adi-N-acetylcystine amide; and separating dried di-N-acetylcystinedimethylester into N-acetylcysteine amide with dithiothreitol,triethylamine and an alcohol.
 2. The process of claim 1, wherein thealcohol is an organic alcohol selected from an alkyl alcohol, methanol,ethanol, propanol, iso-propanol or butanol.
 3. The process of claim 1,wherein the step of contacting L-cysteine with an alcohol and achlorinating reagent to form an organic solution containing L-cystinedimethylester dihydrochloride is conducted at −10 to 10° C. and thenheated to reflux at 65 to 70° C. to completion.
 4. The process of claim1, wherein the chlorinating agent is thionyl chloride.
 5. The process ofclaim 1, further comprising a solvent exchange between the contactingand the combining steps.
 6. The process of claim 1, wherein the step ofcombining is performed at −10 to 10° C.
 7. The process of claim 1,wherein a precipitate formed in the combining step is filtered andwashed with ethyl acetate before drying under vacuum.
 8. The process ofclaim 1, wherein the step of combining uses at least 15 volumesacetonitrile at −10 to 10° C. before adding at least 4 equivalents oftriethylamine followed by at least 2 equivalents of acetic anhydride. 9.The process of claim 1, wherein the organic solvent is ethyl acetate.10. The process of claim 1, further comprising drying the organicsolution removed from the neutralized mixture with a drying agent. 11.The process of claim 1, further comprising the step of free-basing thetriethylamine from the di-N-acetylcystine dimethylester with saturatedsodium bicarbonate after reaction completion.
 12. The process of claim1, wherein the ammonia is provided in the form of aqueous ammoniumhydroxide.
 13. The process of claim 1, wherein the contacting of thedi-N-acetyl-L-cystine ester with ammonia is performed at roomtemperature.
 14. The process of claim 1, wherein no metals are used inthe reduction of di-N-acetylcystine amide into N-acetylcysteine amide.15. The process of claim 1, wherein no trace metal ion impurities arepresent to catalyze oxidation.
 16. The process of claim 1, wherein theremoving the organics under reduced pressure is performed at about 45°C. or less.
 17. The process of claim 1, wherein the removing theorganics under reduced pressure is performed at about 35° C. or less.18. The process of claim 1, wherein the removing the organics underreduced pressure is performed at about 30° C. or less.
 19. The processof claim 1, wherein the removing the organics under reduced pressure isperformed at about 45° C.
 20. The process of claim 1, wherein theorganic solution removed from the neutralized mixture is filtered toremove solids.
 21. The process of claim 1, wherein the one or morereducing agents is selected from tris (2-carboxyethyl)phosphine,thioglycolic acid, dithiothreitol, and the organic solvent is THF ordichloromethane (DCM), isopropanol, or ethanol, and the base istriethylamine.
 22. The process of claim 1, wherein the batch size ofNACA produced under GMP conditions is greater than 1 kg.
 23. The processof claim 1, wherein the batch size of NACA produced under GMP conditionsis greater than 15 kg.
 24. The process of claim 1, wherein the batchsize of NACA produced under GMP conditions is greater than 50 kg. 25.The process of claim 1, wherein the batch size of NACA produced underGMP conditions is greater than 100 kg.
 26. The process of claim 1,wherein the sulfurous odor and taste of NACA is masked by solubilizationin ORA-SWEET/water solution.
 27. The process of claim 1, wherein NACA issuspended in perfluorohexyloctane.
 28. The process of claim 1, whereinNACA is solubilized in phosphate buffer.
 29. A compound having aformula:


30. A process for making N-acetylcysteine amide comprising: A processfor making N-acetylcysteine amide comprising: contacting L-cystine withan alcohol and a chlorinating reagent to form an organic solutioncontaining L-cystine dimethylester dihydrochloride; combining driedL-cystine dimethylester dihydrochloride with a triethylamine, an aceticanhydride, and an acetonitrile to form a di-N-acetylcystinedimethylester; mixing dried di-N-acetylcystine dimethylester withammonium hydroxide to form a di-N-acetylcystine amide; and reducingdried di-N-acetylcystine dimethylester into N-acetylcysteine amide withdithiothreitol, triethylamine, and an alcohol, wherein the reduction iswithout the presence of a metal.
 31. In yet another embodiment, thepresent invention includes a process for making N-acetylcysteine amidecomprising: